The long-term goal of my research program is to understand the molecular mechanisms that allow certain plant and bacterial toxins to cross the endoplasmic reticulum (ER) membrane and enter the cytosol of an intoxicated eukaryotic cell. One such toxin, cholera toxin (CT), is responsible for the life-threatening watery diarrhea of cholera. CT is internalized by target cells and delivered to the ER by retrograde vesicular transport. The catalytic CTA1 polypeptide then crosses the ER membrane, enters the cytosol, and initiates the major toxic effects of CT. The ER-to-cytosol translocation of CTA1 involves the mechanism of ER- associated degradation (ERAD), a quality control system that recognizes misfolded proteins in the ER and exports them to the cytosol for ubiquitination and degradation by the 26S proteasome. The C-terminal hydrophobic region of CTA1 is thought to trigger ERAD activity and stimulate CTA1 translocation to the cytosol; degradation in the cytosol is presumably avoided because CTA1 has a paucity of the lysine residues that serve as ubiquitin attachment sites. Our work has shown that the C-terminal region of CTA1 is not required for toxin entry into the cytosol and that the translocated pool of CTA1 is degraded by a temperature-sensitive, ubiquitin-independent proteasomal mechanism. This degradative mechanism may involve the core 20S proteasome, in contrast to the standard route of ubiquitin-dependent degradation by the 26S proteasome. Both the translocation and degradation of CTA1 may be linked to the heat-labile nature of the isolated CTA1 polypeptide. We believe thermal instability in the CTA1 polypeptide generates a partially unfolded conformational state at 37 degrees C that triggers ERAD activity and renders the cytosolic pool of toxin susceptible to degradation by the 20S proteasome. This model will be tested with experimental conditions involving low pH buffers (pH approximately 6.0) that apparently inhibit the thermal denaturation of CTA1. We predict acidic pH will stabilize the structure of CTA1 and thereby inhibit CTA1 translocation/degradation. Our results will form the basis of a new model for toxin-ERAD interactions with applications to toxin pathogenesis and the development of novel anti-toxin biodefense strategies. [unreadable] Cholera toxin will not function if it cannot enter target cells. Acid-induced changes to the structure of cholera toxin may prevent its entry into target cells and would thus generate resistance to the disease cholera. [unreadable] [unreadable] [unreadable]